1. Field of the Invention
This invention relates to the study of materials using high energy photons and, more particularly, this invention relates to an on-axis method for studying samples using synchrotron-produced x-rays.
2. Background of the Invention
Macromolecular crystallography involves the study of crystal structure of target materials by observing how those structures diffract and otherwise affect incident electromagnetic radiation. These studies are optimized by accurate placement of the target crystal in the line of travel of the radiation.
High-throughput data collection for macromolecular crystallography requires an automated sample mounting and alignment system for crystals that functions reliably when integrated into protein-crystallography beamlines at synchrotrons. Rapid mounting and dismounting of the samples increases the efficiency of the crystal screening and data collection processes, where many crystals can be tested for the quality of diffraction.
A key factor for high performance X-ray protein crystallography beam lines is the overall duty cycle of the beam line including efficient optics alignment, automation of sample handling, crystal visualization and alignment, data collection and data analysis. Many synchrotron radiation facilities are now concentrating on these high-throughput projects, which will have an enormous influence on the overall throughput of the field.
Indirect Image
Technologies
It is normally assumed that sample visualization cannot be done during data collection along the x-ray beam without degradation of the image quality, in conjunction with “kappa geometry” (i.e rotation of a sample around three independent axes) and in the presence of a cold stream such as boiled-off gas from liquid nitrogen or liquid helium. Heretofore, these processes required a microscope with a long working distance also having an optical magnification of about 10×. Also the presence of a beam passage bore in the optics degrades the image because paraxial rays are lost.
On-axis visualization can be done with removable optical components prior to (or after) data collection by swinging a microscope in place. The microscope is used initially to align the crystal. Afterwards, the components of the microscope are retracted from the on-axis area near the sample. Data collection in this configuration relies on stability of the goniostat supporting the sample, stability of the x-ray beam, and precision of the initial alignment. This beam visualization method has a major limitation in that visualization is not done during data collection. Moreover, these machinations require precise maneuvering, and therefore high mechanical stability
Off axis, or “indirect alignment” visualization methods rely on visualization techniques based on microscopes with fixed optical components. However, off axis alignment suffers from parallax error which results in inferior precision of alignment. The best practice with off axis alignment requires two cameras with orthogonal views of the sample.
Typical indirect crystal centering methods comprise two steps. In this method, the visible light cameras are mounted and moving with goniostat support (support of omega, kappa, and sample motions, see #58 infra). First the center of omega rotation of the goniostat is determined by using well defined pointy object (such as AFM cantilever), a fiducially marked cross-hair is then placed at the center of omega rotation in video stream from cameras. Second, the location of the x-ray beam is determined by having the beam strike a phosphor screen; placed at omega center of rotation; a goniostat support is moved so that the fiducially marked cross-hair is at the beam location. Finally, with the help of a visible light microscope, the target crystal is placed manually in the center of omega rotation and at the cross-hairs. Visible illumination is typically done from the side or behind the crystal (bright field illumination).
Other approaches include using high power broadband sources and filtered out UV component to excite the visible fluorescence. Drawbacks to this approach include the need for a large UV source system, and a lack of understanding of the physics involved.
Still others have used pulsed UV (266 nm) lasers. However, such pulsed lasers induce crystal damage due to adiabatic processes. Also, these systems use visible spectrum for imaging with visible light cameras.
Direct Visualization
Technologies
On-axis observation of crystals allows visualization of x-ray beams without parallax distortion and visualization of the crystal from the x-ray beam point of view. Sample alignment with on-axis visualization compares favorably to prior (non-axial) alignment techniques which generally suffer from parallax errors. Misjudgments of fluorescence depth from phosphor illumination at various photon energies can be one of the reasons for misalignment. On-axis visualization allows alignment verification during data collection.
All of the aforementioned off-axis techniques fail to readily locate a biological crystal in the sample holder so as to place it in the center of the x-ray beam. Macromolecular Crystallography at third-generation synchrotrons has been relying primarily on light in the visible spectrum (400 nm-600 nm wavelength) for sample alignment, with optical microscopes being used to achieve sample alignment.
The main motivation for the use of crystal visualization microscopy at the synchrotron beamline is to achieve precise placement of a small biological crystal in the same x-ray beam that is used for measurement of the sample properties. The typical 10 μm-50 μm size biological crystal must be placed at the center of the experimental apparatus, and the sample must be able to rotate around its axis and around the beam axis. Moreover, the x-ray beam center should pass through the center of rotation of the apparatus and through the crystal center. For optimal x-ray diffraction by the sample crystal, a uniform intensity x-ray beam should match the size of the crystal. The typical x-ray beam heretofore used has had a 25-75 μm rectangular cross-section.
The few successful on-axis sample visualization systems in use are devices with line-of-sight view along x-ray beam (from source direction) and without parallax errors (“direct alignment”) visualization. The MD2 diffractometer (Maatel) has on-axis visualization with a compound objective lens (with as many as 10 component lenses) placed very close to the sample (one inch or so) followed by a 45° mirror and finally a camera. The objective lens has a bore drilled through it to allow unobstructed passage of x-rays through the lens. The system introduced by Owens et al. (J. Synchrotron Rad. 16, 173-182 (2009)) utilizes a reflecting telescope with an objective in close proximity to the sample (approximately an inch) a 45° mirror between the objective and the primary mirror and finally a camera. A bore is required through the objective and the 45° mirror to allow passage of the beam.
The disadvantage of current on-axis visualization systems is that paraxial rays that could be used in image formation are lost in the bore drilled through objective and 45° mirror optics. This loss of image-forming paraxial rays results in inferior image quality at the center of the microscope. Also, lenses introduce chromatic aberration (different focal length for different wavelengths) making such a system unsuitable for work spanning a wide range of wavelengths with high image quality at the center needed for beam and sample visualization. The close proximity between the objective and the sample introduces spherical aberration. Moreover, alignment of the apparatus is critical in that one must ensure that the x-ray beam does not impact the walls of the bore through which it passes. Finally the lack of adequate working space near the sample limits the experiments that can be performed. Specifically, one cannot perform the full complement of sample rotations that are necessary for a complete determination of a sample.
A need exists in the art for a sample visualization system that pinpoints location of the sample relative to the radiation beam used to illuminate the sample. The system should also facilitate sample visualization during data gathering. The system should expedite alignment of the sample to enhance streamlining of such processes.